1. Novel approach to identify phenoloxidases inhibitors: Optimization of spectrophotometric MBTH assay for high throughput use enzymatic assays and analysis
- Author
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Charlotte Himber, Christophe Flahaut, Thierry Grard, Cédric Le Bris, Benoit Cudennec, Pascal Dhulster, Estelle Zeyer, Vincent Phalip, Guillaume Duflos, Egon Heuson, Anne Brisabois, Institut Charles Viollette (ICV) - EA 7394 (ICV), Université d'Artois (UA)-Institut National de la Recherche Agronomique (INRA)-Université du Littoral Côte d'Opale (ULCO)-Institut Supérieur d'Agriculture-Université de Lille, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Université de Lille, and Université d'Artois (UA)
- Subjects
hydrochloride ,Hydrazone ,01 natural sciences ,chemistry.chemical_compound ,0404 agricultural biotechnology ,Sulfite ,[SDV.IDA]Life Sciences [q-bio]/Food engineering ,Phenol ,High throughput screening assay development ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,IC50 ,Detection limit ,chemistry.chemical_classification ,Oxidase test ,Chromatography ,Spectrophotometric ,010401 analytical chemistry ,3-Methyl-2-benzothiazolinone hydrazone ,04 agricultural and veterinary sciences ,Glutathione ,Phenoloxidase inhibition ,040401 food science ,0104 chemical sciences ,Dissociation constant ,Penaeus monodon ,chemistry ,Post-mortem melanosis ,Food Science ,Biotechnology - Abstract
International audience; This study presents a miniaturized 3-methyl-2-benzothiazolinone hydrazone hydrochloride-assay (MBTHassay) 1 , optimized for high-throughput measurement of shrimp phenol oxidase activities and the screening of potential inhibitors of post-mortem melanosis. To validate this approach, assay parameters, including limit of detection and dynamic range, were compared to values obtained for DOPAchrome-assay, used here as a reference method. A reliable high-throughput automatized analysis was also performed, under standardized parameters, in order to compare and characterize 40 potential inhibitors of phenol oxidase activities. Among the selected molecules, twenty showed inhibitory action. Enzyme-inhibitor dissociation constant (K i), median inhibitor concentration (IC 50) and inhibition modes could be determined for nine of them. 4-Hexylresorcinol, Nphenylthiourea L-cystein and glutathione displayed K i values lower than sulfite salts (Na 2 S 2 O 5 and Na 2 SO 3) used as positive controls.
- Published
- 2018